The blotting of electrophoretically separated biological molecules onto solid supports has been used for a variety of purposes, including to perform immunoassays of the blotted molecules. The "Western blot" is perhaps one of the best known approaches in this respect, which allows the identification of separated and then blotted proteins by the use of antibody probes of the solid support. See generally, Renart, J. and I. V. Sandoval, "Western Blots", Methods in Enzymol., Chapt. 33, Vol. 104, Acad. Press. (1984).
In addition to proteins and polysaccharides, lipopolysaccharides have also been identified by transfer techniques. See, e.g., Pyle, S. W. and W. B. Schill, "Rapid Serological Analysis of Bacterial Lipopolysaccharides by Electrotransfer to Nitrocellulose", J. Immunol. Meth., 85:371-382 (1985).
Another technique, known as "colony blotting", generally involves the transfer of biological molecules from the surface of a culture plate, such as an agar plate having bacterial colonies on its surface, to a solid support. See, for instance, Kemp, D. J. and A. F. Cowman, "Detection of Expressed Polypeptides by Direct Immunoassay of Colonies", in Methods in Enzymol., Chapt. 83, Vol. 79, Acad. Press. (1981), describing a process in which the colonies are first lysed. The proteins released from the lysed cells are then bound covalently to CNBr paper and detected by reaction with radiolabeled antibodies.
Colony blotting is not generally used with pour plate type culture media, where a majority of the colonies are not accessible at the surface of the media, nor has it been described previously for use with reconstitutable dry culture media such as those described more fully below.
By virtue of the time, effort, expertise, and cost required, immunochemical identification of microbes as described above is typically used in the context of the research laboratory as opposed to its use for routine high volume screening of samples for the presence of microbes such as screening done in the food, cosmetic, and drug industries, or for environmental purposes.
In contrast, such routine, high volume screening of samples for the presence of microbes is still typically carried out by laborious and/or relatively insensitive biochemical or microbiological techniques. These techniques often involve whole cell counts and/or the use of preenrichment broth culture followed by selective agar plates. Such techniques generally result in either a simple quantitative answer regarding the total microbial count, or a qualitative (e.g., positive/negative) answer regarding the presence of a particular microbial species of interest. Current rapid screening techniques are rarely if ever able to provide both an indication of the presence of a particular microbe as well as an indication of the concentration of the microbe, let alone allow the user to then locate and isolate the identified microbe, e.g., for further growth and characterization.
A good example highlighting the need for better screening methods is that of the prevalence in meat samples of the pathogenic E. coli strain identified as "O157:H7". See, e.g., Riley, "The Epidemiological, Clinical, and Microbiological Features of Hemorrhagic Colitis", Ann. Rev. Microbiol. 41:383-407 (1987), the disclosure of which is incorporated herein by reference. Among the tests described as useful for the detection of E. coli O157:H7 are those described by Doyle et al., Applied and Environmental Microbiology, 53(10):2394-2396 (1987), and Todd et al., Applied and Environmental Microbiology, 54(10):2536-2540 (1988), the disclosure of each of which are incorporated herein by reference.
The meat industry currently relies, in large part, on the method of Doyle cited above. This method is basically an enrichment procedure in which samples are grown in a preenrichment broth, diluted, and grown on selective agar in an attempt to visually identify E. coli O157:H7 colonies from among the colonies of non-pathogenic background cells. This method suffers from a number of drawbacks, including the need to visually distinguish colonies of E. coli O157:H7 from other colonies. Given the typically large number of non-pathogenic bacteria present it is very possible that E. coli O157:H7 cells are missed, thereby leading to an undesirably high number of false negatives.
In view of the fact that the U.S. Food and Drug Administration ("FDA") and others are becoming increasingly concerned with the need to identify and quantitate microbial pathogens in samples such as food, combined with the fact that food industry needs a method that is quick, sensitive, cost effective, and practical, the presently available research-oriented techniques are not suitable for such purposes.